The mechanism of carrier-mediated transport of folates in BeWo cells: the involvement of heme carrier protein 1 in placental folate transport

The aim of this study was to elucidate the mechanism of folate transport in the placenta. A study of folate was carried out to determine which carriers transport folates in the human choriocarcinoma cell line BeWo, a model cell line for the placenta. We investigated the effects of buffer pH and various compounds on folate uptake. In the first part of the study, the expression levels of the mRNA of the folate receptor alpha (FRalpha), the reduced folate carrier (RFC), and heme carrier protein 1 (HCP1) were determined in BeWo cells by RT-PCR analysis. Folate uptake into BeWo cells was greater under an acidic buffer condition than under a neutral one. Structure analogs of folates inhibited folate uptake under all buffer pH conditions, but anion drugs (e.g., pravastatin) inhibited folate uptake only under an acidic buffer condition. Although thiamine pyrophosphate (TPP), a substrate of RFC, had no effect on folate uptake, hemin (a weak inhibitor of folate uptake via HCP1) decreased folate uptake to about 80% of the control level under an acidic buffer condition. Furthermore, kinetic analysis showed that hemin inhibited the low-affinity phase of folate uptake under an acidic buffer condition. We conclude that pH-dependent folate uptake in BeWo cells is mediated by at least two carriers. RFC is not involved in folate uptake, but FRalpha (high affinity phase) and HCP1 (low affinity phase) transport folate in BeWo cells.     

A new apparatus for CT-guided stereotactic surgery

Combining whole-body CT scan with a stereotactic system, the authors have developed and applied clinically an apparatus which readily provides intraoperative CT images, making it possible to confirm the location of the target point and ascertain the intraoperative environment. It takes about 9 s to obtain a CT image. Our purpose is to make stereotactic surgery, a kind of blind surgery, as safe and reliable as a visualized procedure by intraoperative CT scanning. By the method, in which there is very little invasion under local anesthesia, evacuation of deep-seated intracerebral hematomas as well as brain abscesses and also biopsy or brachytherapy of brain tumors in the brain can be done with safety and reliability.     

Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells

Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest-derived ectomesenchyme follows its specific genetic control. The rat small eye ( rSey ) involves a mutation in the Pax-6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno-orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax-6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild-type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild-type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.     

Intestinal uptake of nateglinide by an intestinal fluorescein transporter

Nateglinide, a novel oral hypoglycemic agent, rapidly reaches its maximum serum concentration after oral administration, suggesting that it is rapidly absorbed in the intestine. However, nateglinide itself is not transported by MCT1 or PEPT1. The aim of this study was to characterize the transporters on the apical side of the small intestine that are responsible for the rapid absorption of nateglinide. It has been reported that the uptake of fluorescein by Caco-2 cells occurs via an H+-driven transporter and that the intestinal fluorescein transporter is probably not MCT1. We examined the contribution of the fluorescein transporter to the uptake of nateglinide by Caco-2 cells. Fluorescein competitively inhibited H+-dependent nateglinide uptake. All of fluorescein transporter inhibitors examined reduced the uptake of nateglinide. Furthermore, nateglinide inhibited fluorescein uptake. We conclude that the intestinal nateglinide/H+ cotransport system is identical to the intestinal fluorescein/H+ cotransport system.     

Uptake dynamics of scrapie agent in the intestinal villous epithelium of suckling and weanling Syrian hamsters

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.     

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages. Thus, the new ES cell line expressing EGFP ubiquitously is useful for basic research in the field of regenerative medicine. The B6G-2 cell line is freely available from the BioResource Center, RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Methyl green-pyronin stain distinguishes proliferating from differentiated nonproliferating cell nuclei after acid denaturation of DNA

We applied methyl green-pyronin (MG-P) stain, which is usually used for the selective staining of DNA and RNA, to frozen sections of rat jejunal and esophageal mucosa, following digestion with RNase and treatment with various concentrations of HCl. The pyroninophilia of the nuclei increased with increasing strength of the acid, but the susceptibility of the nuclei to acid differed among cell populations. In the jejunal epithelium, at an appropriate acid strength the nuclei in the crypts of Lieberkuhn were less acid-sensitive and remained blue-green, whereas those in the villi were more pyroninophilic and stained lavender. Under the same conditions, the nuclei in the basal layer of the esophageal epithelium were blue-green and those in the spinous and granular layers were increasingly lavender. These results suggest that in cell-renewal systems the differentiated, nonproliferating cells are more sensitive to acid denaturation of DNA than the undifferentiated, actively proliferating cells. MG-P stain, which is able to distinguish double-stranded from single-stranded DNA, may be used as a tool to stain proliferating and nonproliferating cell nuclei differentially in tissue sections.     

Quantification of intracellular and extracellular prostanoids stimulated by A23187 by liquid chromatography/electrospray ionization tandem mass spectrometry

Prostanoids are bioactive substances that contribute to various biological and pathological processes. To evaluate both extracellular and intracellular levels of prostanoids at the same time, we developed methods for quantification of extracellular and intracellular levels of prostanoids, including prostaglandin E(2) (PGE(2)), PGD(2), PGF(2α), 6-keto PGF(1α), and TXB(2), in cultured cells using liquid chromatography/tandem mass spectrometry (LC/MS/MS), and we validated the LC/MS/MS methods. A solid-phase extraction cartridge was used for extraction of prostanoids. The prostanoids were separated by a C(18) column with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). Calibration curves of extracellular measurement for the prostanoids were linear in the range from 0.1 to 100 ng/mL (r(2)>0.999), and those of intracellular measurement were linear in the range from 0.05 to 50 ng (r(2)>0.999). Validation assessment showed that both methods of extracellular and intracellular measurements were highly reliable with good accuracy and precision. We also applied the methods to human airway epithelial Calu-3 cells and human lung adenocarcinoma epithelial A549 cells.